Atomic force microscopes (AFMs) are capable of producing images at molecular resolutions in water, making them a useful tool for biological and chemical imaging. AFMs, however, are limited because when complex samples are imaged, it is nearly impossible to differentiate between molecular targets, such as proteins, of the same molecular weight from the topographical image alone.
Recognition imaging is a technique that gives an AFM chemical sensitivity. U.S. Pat. No. 7,152,462, which is herein incorporated by reference in its entirety, discloses an atomic force microscope having an antibody tethered to the probe tip. The antibody tethered to an oscillating AFM sensing probe, binds to its antigen and changes the pattern of oscillation as the probe is scanned over the surface. A map of these changes, superimposed onto the topographic image, can show where the targets are located in the image.
FIGS. 1a and 1b show a typical prior art AFM configured for recognition imaging. The AFM's sensing probe consists of an oscillating cantilever 101 and a sensing agent 102 tethered to the probe's tip 107 by a short, flexible tether 108. The AFM contains a mechanism for recording the oscillation waveform detected as the probe is scanned across a surface 103. If the sensing agent 102 does not bind to a target on the surface 103, the waveform can look like the graph oscillation pattern 104 shown above FIG. 1a. If the sensing agent 102 does bind to a target on the surface 103, the waveform might look like the graph oscillation pattern 106 shown above FIG. 1b. 
While a configuration such as the one shown in FIGS. 1a and 1b can provide a means for mapping compositions such as proteins on a nm-scale, it is limited to detecting only one target per image. A critical aspect of many chemical and biological systems lies in the relative spatial arrangement of different participants in the reactions or processes. Many biological pathways involve complex and so-called associated “co-factors,” such as other proteins that must be recruited before an assembly becomes active. For example, in the case of a complex process like initiation of gene transcription, many factors must be recruited and assembled. Therefore, it is often important to know not only what factors are present individually in a sample, but also to know where one factor is positioned with respect to another.
In order to locate a different target using the systems and methods currently available in the art, the user must change the probe tip to one having a different sensing agent and then record a second image. Because of the time it takes to change the probe tip as well as the process of physically moving the tip, it is difficult, if not impossible, to draw any conclusions as to the relative locations of different targets based solely on the two recorded images. Statistical analysis of the two images can produce estimates as to the targets' relative positions, but such analysis is computationally complex and still only produces estimated results.
Therefore, there is a need in the art of recognition imaging for a probe that enables the determination of the relative locations of at least two different factors at once.